The most common manipulation of vectors in molecular biology laboratories is the transfer of a gene of interest into a vector of choice. The resulting recombinant vectors allow specialized applications such as expression in mammalian cells, expression in bacterial hosts, purification of the native protein through employment of specialized (vector provided) purification tags or detection of interaction with other proteins (two-hybrid systems). Typically, cloning is achieved through restriction digestion, isolation of the desired fragments and reconstitution of the desired plasmid by ligation. Although this technique has been routinely employed for approximately 20 years, it is still error-prone and cumbersome.
There is a need in the art for a method of transferring a desired coding region to a vector of interest without the use of restriction enzyme recognition sites and restriction enzymes. In prior art methods, multiple restriction enzymes are employed for the removal of the desired coding region and the reaction conditions used for each enzyme may differ such that it is necessary to perform the excision reactions in separate steps. In addition, it may be necessary to remove a particular enzyme used in an initial restriction enzyme reaction prior to completing all restriction enzyme digestions. This requires a time-consuming purification of the subcloning intermediate. More recently, recombinase-based cloning methods have been developed. However, the current methods require multiple recombination events.
There is a need in the art for cloning of newly discovered sequences, such as new genes. Thus there is a need in the art for more efficient techniques for transfer of the genes of interest into a vector of choice. It is desirable that such a technique permits high fidelity, high efficiency and a minimum number of handling steps to allow adaptation to automated procedures.
There is a need in the art for a method for the cloning of a DNA molecule which permits rapid transfer of the DNA molecules from one vector to another without the need to rely upon restriction enzyme digestions.
The present invention provides a method of transfer of a gene of interest to a product vector comprising: contacting in vitro (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) a site-specific recombinase, wherein the contacting permits formation of a co-integrate vector comprising the first and the second vector. The co-integrate vector is subsequently introduced into a prokaryotic host cell so as to permit the formation of a product vector comprising the gene of interest interposed between the double-stranded origin of replication of the second vector and the site-specific recombination recognition site, the single-stranded origin of replication of the second vector, and the gene encoding the second selectable marker, wherein the product vector does not include both of the gene encoding the negative selectable marker and the gene encoding the first selectable marker.
The present invention further provides a method of transfer of a gene of interest to a co-integrate vector comprising contacting in vitro (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon; and (c) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) a site-specific recombinase, wherein the contacting permits formation of a co-integrate vector comprising the first and the second vector.
In one embodiment, the co-integrate vector is introduced into a prokaryotic host cell.
The present invention further provides a method of transfer of a gene of interest to a product vector comprising introducing into a prokaryotic host cell which expresses a gene encoding a site-specific recombinase (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon; and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site, and wherein said prokaryotic host cell further expresses a gene encoding a rep protein which can initiate replication at the double stranded origin of replication. The introduction of the first and second vector to the prokaryotic host cell permits formation of a product vector comprising the gene of interest interposed between the double-stranded origin of replication of the second vector and the site-specific recombination recognition site, the single-stranded origin of replication of the second vector, and the gene encoding the second selectable marker, the product vector not including both of the negative selectable marker and the gene encoding the first selectable marker.
The present invention further provides a method of transfer of a gene of interest to a co-integrate vector comprising introducing into a prokaryotic host cell which expresses a gene encoding a site-specific recombinase a first vector and a second vector so as to permit recombination of the first and second vectors to produce a co-integrate vector, wherein the first vector comprises (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and the second vector comprises (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site.
In one embodiment the introduction of the first and second vector to the host cell permits formation of a product vector comprising the gene of interest interposed between the double-stranded origin of replication of the second vector and the site-specific recombination recognition site, the single-stranded origin of replication of the second vector, and the gene encoding the second selectable marker, wherein said host cell expresses a gene encoding a rep protein which can initiate replication at the double stranded origin of replication of the first and second vector. The product vector does not include both of the negative selectable marker and the gene encoding the first selectable marker.
In a preferred embodiment, the prokaryotic host cell is grown under conditions which permit the first and second vectors to recombine to form a co-integrate vector.
In a further embodiment, following introduction of either the first and second vectors, or the co-integrate vector into the prokaryotic host cell, the product vector is isolated from the host cell.
In a still further embodiment, the first and second selectable markers are different.
In one embodiment, the site-specific recombinase recognition site is selected from the group consisting of: loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxxcex9486, loxxcex94117, frt, dif, Kw, xcex-att, and "PHgr"C31 att sites.
In one embodiment, the double-stranded origin of replication is the double-stranded origin of replication of the filamentous bacteriophage f1.
In a further embodiment, the double-stranded origin of replication is the double-stranded origin of replication of the plasmid pKym.
In one embodiment, the negative selectable marker is one of rpsL and sacB.
In one embodiment, the gene encoding one of the first or second selectable marker, independently, is selected from the group consisting of: kanarnycin resistance gene, the ampicillin resistance gene, the tetracycline resistance gene, the chloramphenicol resistance gene, spectinomycin resistance gene, gentamycin resistance gene, and the streptomycin resistance gene.
The present invention further provides a vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication, (c) a site-specific recombination recognition site, and (d) a gene encoding a selectable marker, wherein the negative selectable marker is interposed between the double-stranded origin of replication and the site-specific recombination recognition site.
The invention still further provides a pair of vectors comprising a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site.
The present invention also provides a product vector comprising (a) a gene of interest, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a nucleic acid sequence encoding a second selectable marker, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site, and wherein the vector does not include both of the gene encoding the negative selectable marker and the gene encoding the first selectable marker.
In addition, the present invention provides a kit for the transfer of a gene of interest to a product vector comprising (1) a first vector comprising (a) a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (d) a site-specific recombination recognition site, wherein the gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the gene encoding the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) packaging materials therefore.
The invention still further provides a kit for the transfer of a gene of interest to a product vector comprising (1) a first vector comprising (a) a cloning site for insertion of a gene of interest, (b) a gene encoding a first selectable marker, (c) a double-stranded origin of replication of a rolling circle replicon, and (c) a site-specific recombination recognition site, wherein the cloning site for insertion of a gene of interest is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (2) a second vector comprising (a) a negative selectable marker, (b) a double-stranded origin of replication of a rolling circle replicon, (c) a site-specific recombination recognition site, (d) a single-stranded origin of replication, and (e) a gene encoding a second selectable marker, wherein the negative selectable marker is interposed between the double-stranded origin of replication of a rolling circle replicon and the site-specific recombination recognition site; and (3) packaging materials therefore.
In one embodiment, the kit further comprises a host cell capable of supporting a rolling circle double-stranded origin of replication.
In a further embodiment, the kit further comprises a site-specific recombinase.
In a still further embodiment, the kit comprises a host cell comprising a site-specific recombinase specific for the site-specific recombination site.
In a still further embodiment of the invention, the host cell is transfectible.
As used herein, xe2x80x9cinterposedxe2x80x9d refers to a nucleic acid molecule which has, immediately adjacent to its 5xe2x80x2 most end, either a double-stranded origin of replication of a rolling circle replicon or a site-specific recombination recognition site, and has, immediately adjacent to its 3xe2x80x2 most end whichever of the double-stranded origin of replication of a rolling circle replicon or site-specific recombination recognition site that is not immediately adjacent to the 5xe2x80x2 most end. As used herein, xe2x80x9cimmediately adjacentxe2x80x9d means that there are between 0 and 500 nucleotides between the 5xe2x80x2 end of the nucleic acid molecule and the 3xe2x80x2 nucleotide of a sequence consisting of either a double-stranded origin of replication of a rolling circle replicon or a site-specific recombination recognition site, and between 0 and 500 nucleotides between the 3xe2x80x2 end of the nucleic acid molecule and the 5xe2x80x2 nucleotide of a sequence consisting of whichever of the a double-stranded origin of replication of a rolling circle replicon or site-specific recombination recognition site is not adjacent to the 5xe2x80x2 end of the nucleic acid molecule.
As used herein, xe2x80x9cdouble-stranded origin of replication of a rolling circle repliconxe2x80x9d refers to a nucleic acid sequence which contains the physical and functional elements required in cis for the initiation of the first strand synthesis. A xe2x80x9cdouble-stranded origin of replication of a rolling circle repliconxe2x80x9d may be isolated from plasmids of both gram-positive and gram-negative bacteria, bacteriophage or any organism which can support replication by a rolling circle mechanism. Such organisms include, but are not limited to Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Streptomyces, Actinobacillus actinobycetemcomitans, Bacteroides, cyanobacteria, Helobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, or Treponema denticola, Bacillus thuringiensis, Staphlococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Streptococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, Streptomyces ghanaenis, Escherichia coli, Halobacterium strain GRB, and Halobaferax sp. strain Aa2.2. Examples of plasmids which possess a xe2x80x9cdouble-stranded origin of replication of a rolling circle repliconxe2x80x9d useful in the present invention include, but are not limited to the following: pT181, pC221, pC223, pCW7, pHD2, pLUG10, pOg32, pS194, pT127, pTZ12, pUB112, pE194, pA1, pC1305, pCI411, pFX2, pKMK1, pLS1, pSH71, pWV01, pC194, pAMxcex11, pA, pPL, pSSU1, p1414, pDC123, pBAA1, pBC1, pBC16, pBP614, pBS2, pC300, pCA2.4, pCB101, pCB2.4, pCC5.2, pFTB14, pGT5, pJDB21, pKYM, pLAB1000, pLot3, pLP1, pOX6, pRF1, pRBH1, pSH1415, pSN1981, pTA1060, pTD1, pTHT15, pUB110, pUH1, pVA380-1, pWC1, pEGB32, p353-2, pSN2, pBI143, pE5, pE12, pIM13, pNE131, pT48, pTCS1, pZMO2, pIJ101, pBL1, pJV1, pSG5, pSN22, pC1305, pG12, pGRB1, pHK2, pHPK255, pTX14-1, pTX14-3, or pVT736-1.
As used herein, a xe2x80x9csingle-stranded origin of replicationxe2x80x9d refers to a nucleic acid sequence at which replication of single-stranded DNA is initiated. A xe2x80x9csingle-stranded origin of replicationxe2x80x9d is strand and orientation specific and must be present in a single-stranded form to actively initiate replication. A xe2x80x9csingle-stranded origin of replicationxe2x80x9d useful in the present invention may include any single-stranded origin of replication known to those of skill in the art, or may be selected from ssos, ssoA, ssoT, ssoW, ssoU types of single-stranded origins of replication, or may be selected from the single-stranded origins of replication present in the following plasmids: pT181, pC221, pC223, pCW7, pHD2, pLUG10, pOg32, pS194, pT127, pTZ12, pUB112, pE194, pA1, pC1305, pCI411, pFX2, pKMK1, pLS1, pSH71, pWV01, pC194, pAMxcex11, pBAA1, pBC1, pBC16, pBP614, pBS2, pA, pPL, pSSU1, p1414, pDC123, pC300, pCA2.4, pCB101, pCB2.4, pCC5.2, pFTB14, pGT5, pJDB21, pKYM, pLAB1000, pLot3, pLP1, pOX6, pRF1, pRBH1, pSH1451, pSN1981, pTA1060, pTD1, pTHT15, pUB110, pUH1, pVA380-1, pWC1, pEGB32, p353-2, pSN2, pBI143, pE5, pE12, pIM13, pNE131, pT48, pTCS1, pZMO2, pIJ101, pBL1, pJV1, pSG5, pSN22, pC1305, pG12, pGRB1, pHK2, pHPK255, pTX14-1, pTX14-3, PCR-ScriptAmpSK+, filamentous phage (f1), "PHgr"X174, pB#322, or pVT736-1
As used herein, xe2x80x9crolling circle replicationxe2x80x9d refers to a mode of replication utilized by some DNA molecules including certain bacteriophage genomes and also found in Xenopus oocytes during amplification of extrachromosomal ribosomal DNA. DNA synthesis initiates at a double-stranded origin of replication from which a sole replication fork proceeds around the template nucleic acid. As the fork revolves, the newly synthesized strand displaces the previously synthesized strand from the template, producing a characteristic tail comprised of single-stranded DNA. The displaced strand is released from the plasmid once the replication fork encounters the double-stranded origin of replication, recircularized and may then be made double-stranded via DNA synthesis which initiates from the single-stranded origin of replication and processed into single or multimeric copies of the original DNA.
As used herein, a xe2x80x9csite-specific recombinasexe2x80x9d refers to an enzyme that binds a specific DNA recognition sequence within a first DNA molecule and, upon forming a protein DNA complex at this specific recognition site, promotes strand exchange with a second protein DNA complex which includes a second molecule of the same xe2x80x9csite-specific recombinasexe2x80x9d bound to a different site on the first DNA molecule or a second DNA molecule having the same recognition sequence, recombining the first and second DNA sequences adjacent to each recombinase recognition site to form a recombined DNA which includes sequences of both the first and second DNA molecules.
As used herein, a xe2x80x9csite-specific recombination recognition sitexe2x80x9d refers to a nucleic acid sequence (i.e., site) which is recognized by a sequence-specific recombinase and which becomes, or is adjacent to the crossover region during the site-specific recombination event. Examples of site-specific recombination sites include, but are not limited to loxP, loxP2, loxP3, loxP23, loxP511, loxB, loxC2, loxL, loxR, loxxcex9486, or loxxcex94117 sites, frt sites, "PHgr"C31 att sites, Kw sites, and dif sites.
As used herein, xe2x80x9cvectorxe2x80x9d refers to a nucleic acid molecule that is able to replicate in a host cell. A xe2x80x9cvectorxe2x80x9d is also a xe2x80x9cnucleic acid constructxe2x80x9d. The terms xe2x80x9cvectorxe2x80x9d or xe2x80x9cnucleic acid constructxe2x80x9d includes circular nucleic acid constructs such as plasmid constructs, cosmid vectors, etc. as well as linear nucleic acid constructs (e.g., PCR products, N15 based linear plasmids form E. coli). The nucleic acid construct may comprise expression signals such as a promoter and/or enhancer (in such a case it is referred to as an expression vector). Alternatively, a xe2x80x9cvectorxe2x80x9d useful in the present invention can refer to an exogenous nucleic acid molecule which is integrated in the host chromosome, providing that the integrated nucleic acid molecule, in whole, or in part, can be converted back to an autonomously replicating form.
As used herein, xe2x80x9cselectable markerxe2x80x9d refers to any one of numerous markers which permit selection of a cell containing a vector expressing the marker known in the art. For example, a gene coding for a product which confers antibiotic resistance to the cell, which confers prototrophy to an auxotrophic strain, or which complements a defect of the host. A xe2x80x9cselectable markerxe2x80x9d may be a protein necessary for the survival or growth of a transformed host cell grown in a selective culture medium. Host cells not transformed with the vector containing the selectable marker will not survive in the selective culture medium. Typical selectable markers are proteins that confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, kanamycin, chloramphenicol, streptomycin, spectinomycin, gentamycin, or tetracycline. Alternatively, selectable markers may encode proteins that complement auxotrophic deficiencies or supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. Alternative selectable markers, useful in the present invention are suppressor tRNAs. A number of selectable markers are known to those of skill in the art and are described for instance in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989.
As used herein, a xe2x80x9cnegative selectable markerxe2x80x9d refers to a protein which, when expressed by a host cell confers susceptibility of that host cell to agents such as one of the selectable markers referred to above, e.g., an antibiotic or toxin. Genes encoding xe2x80x9cnegative selectable markersxe2x80x9d useful in the present invention include, but are not limited to rpsL, sacB, hsv-tk, GLUT-2, or gpt. Alternatively, promoters or other functional elements required for the efficient expression of a negative selectable marker gene also can function as negative selectable markers. Alternatively, a negative selectable marker may be a restriction site, recognized by a host restriction system which would lead to cleavage of a plasmid containing the sequence, thus eliminating the functionality of the plasmid. An additional example of a negative selectable marker, useful in the present invention is the so called kill genes derived from low copy number plasmids such as the Fxe2x80x2 derived ccd gene (Boe et al., 1987 J. Bacteriol 169:4646). Insertion of a xe2x80x9cnegative selectable markerxe2x80x9d into a vector of the present invention would permit one of skill in the art to selectively eliminate that vector.
As used herein, xe2x80x9cintroducingxe2x80x9d refers to the transfer of a nucleic acid molecule from outside a host cell to inside a host cell. Nucleic acid molecules may be xe2x80x9cintroducedxe2x80x9d into a host cell by any means known to those of skill in the art, or taught in numerous laboratory texts and manuals such as Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York (1989). Means of xe2x80x9cintroducingxe2x80x9d nucleic acid into a host cell include, but are not limited to heat shock, calcium phosphate transfection, electroporation, lippofection, and viral mediated gene transfer.
As used herein, a xe2x80x9cprokaryotic host cellxe2x80x9d refers to any organism which can replicate plasmid DNA by a rolling circle mechanism, including, but not limited to gram-positive bacteria, and gram-negative bacteria. Alternatively a xe2x80x9cprokaryotic host cellxe2x80x9d refers to any organism which is capable of supporting replication from a single-stranded origin of replication. As used herein, a xe2x80x9cprokaryotic host cellxe2x80x9d also refers to any organism which is capable of supporting nucleic acid replication from both double- and single-stranded origins of replication. More specifically, a xe2x80x9cprokaryotic host cellxe2x80x9d useful in the present invention may be selected from the group including, but not limited to Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Streptomyces, Actinobacillus actinobycetemcomitans, Bacteroides, cyanobacteria, Escherichia coli, Helobacter pylori, Selnomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, or Treponema denticola, Bacillus thuringiensis, Staphlococcus lugdunensis, Leuconostoc oenos, Corynebacterium xerosis, Lactobacillus plantarum, Streptococcus faecalis, Bacillus coagulans, Bacillus ceretus, Bacillus popillae, Synechocystis strain PCC6803, Bacillus liquefaciens, Pyrococcus abyssi, Selenomonas nominantium, Lactobacillus hilgardii, Streptococcus ferus, Lactobacillus pentosus, Bacteroides fragilis, Staphylococcus epidermidis, Staphylococcus epidermidis, Zymomonas mobilis, Streptomyces phaechromogenes, Streptomyces ghanaenis, Halobacterium strain GRB, and Halobaferax sp. strain Aa2.2.
An advantage of the present invention is that it provides a method for the improved transfer of a gene of interest from one vector to another, without the need for the traditional steps of restriction enzyme digestion, purification, and ligation. A further advantage of the present invention is that it provides a method of transfer of genes of interest into a vector of choice with high fidelity, high efficiency, and a minimal number of handling steps which would allow for the adaptation of the present invention to automated procedures.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims